Synthesis and immunogenicity of rotavirus genes using a baculovirus expression system

ABSTRACT

A method to express rotavirus genes in a baculovirus system. Different clones are used to express rotavirus genes for all of the viral proteins. These proteins are isolated in their native conformation. Some of these proteins show antigenic properties and are used to vaccinate human, agricultural animals and pet animals against diarrheal disease. The antigenic proteins are also used to detect the presence of the viral infectious agent either by themselves or in conjunction with antibodies produced against the antigenic proteins.

This invention was supported in part through grant number NIH AM30144from the National Institutes of Health. The U.S. government may havecertain rights to this invention.

This is a division of application Ser. No. 08/385,993, filed Feb. 9,1995, now abandoned, which is a continuation of application Ser. No.07/830,587, filed Feb. 6, 1992, now abandoned, which is a division ofapplication Ser. No. 06/947,773, filed Dec. 30, 1986, now U.S. Pat. No.5,186,933.

FIELD OF THE INVENTION

The present invention relates generally to a method for expression ofrotavirus genes in a baculovirus system. More specifically, theinvention relates to the expression of rotavirus genes using abaculovirus vector for the production of antigens to be used in thedetection of gastrointestinal disease caused by rotaviruses and in thedevelopment of vaccines against rotavirus infections.

BACKGROUND OF THE INVENTION

The recognition that rotavirus is an important etiologic agent oflife-threatening infantile diarrheal disease has led to significantefforts to control the virus and to prevent the disease. Estes et al.,"Rotavirus Antigens". In Atassi and Backrach eds., Immunobiology ofproteins and peptides-III. Plenam New York p. 201-14 (1985); andKapikian, A.Z., et al. In Fields, B.N., et al. (eds.), Virology, RavenPress, New York, p. 863-906 (1985). Although it is known that oralrehydration is an effective method for reducing diarrheal diseasemortality, other interventions are needed to reduce morbidity andpossibly eradicate this disease. Eradication of the disease wouldrequire immunization on a global population basis. This immunizationcould involve the live attenuated pathogens themselves orpathogen-specific antigenic proteins that induce neutralizing protectionfrom disease (possibly mediated by antibodies). Elucidation andunderstanding of the rotavirus gene structure will greatly facilitatethe efforts to eradicate the disease.

There are several problems associated with efficacy and safety whenusing live rotavirus vaccines:

(1) A vaccine that only produces a "mild" form of the disease may notprovide safe and effective immunization in the gastrointestinal tract.Although viruses are relatively efficient in inducing resistance tosubsequent infection, resistance induced by oral vaccination withattenuated virus strains may show more variability.

(2) There are significant risks that the attenuated virus will revert tovirulence.

(3) Although the live vaccines may be efficious and safe when tested orused in children from developed countries, there is no guarantee thatthe same vaccine will be safe in children in developing countries. Thisis a serious concern since the susceptibility to diarrheal disease isenhanced in children in developing countries due to malnutrition and/orconcurrent infections from other pathogens.

(4) Because of the expense of production and safety testing, the cost ofdistributing live vaccines in developing countries, where they areneeded the most, may be prohibitive.

(5) Finally, in developing countries concurrent infections with multipleenteric pathogens may interfere with vaccine "takes".

The genome of rotavirus consists of 11 segments of double-stranded RNA.The genomic RNA is enclosed within a double-layered protein capsid thatconsists of the structural proteins VP1 to VP9. Estes, M.K. et al,"Rotavirus Antigens". In Atassi and Bachrach eds., Immunobiology ofproteins and peptides-III. Plenum, New York p. 201-14 (1985). Eachgenome segment encodes at least one protein. Chan, W.K., et al.,Virology 151:243-252, (1986), and Mason, B.B., et al., J. Virol,46:413-423, 1983. The outer capsid protein VP3 functions as a viralhemagglutinin and also plays a role in inducing neutralizing antibodies.VP7 is an outer capsid glycoprotein which also induces neutralizationantigen antibodies; VP7 is reportedly the all-attachment protein. Themajor capsid protein, VP6, is located on the inner capsid. This proteincomprises greater than 80% of the protein mass of the viral particle andcontains the subgroup antigen (S antigen VP6). Estes, M.K., et al.,"Rotavirus Antigens" In M.Z. Atassi et al. (eds.) Immunology of Proteinsand Peptides-III, Plenum, New York, pp. 201-214 (1985). The presence ofVP6 on virus particles has been associated with viral polymeraseactivity. Bican, P., et al., J. Virol, 6:1113-1117 (1982). VP6 interactswith viral proteins during replication and assembly. Estes, M.K., etal., "Rotavirus Antigens" In M.Z. Atassi et al. (eds.) Immunology ofProteins and Peptides-III, Plenum, New York, pp 201-214 (1985). Althoughthis interaction is not completely understood, it involves binding toRNA genomic segments or transcripts. VP6 possesses an oligomeric,possibly trimeric, confirmation. Gorziglia, M. C. et al., J. Gen. Virol,66:1889-1900 (1985). Additionally, VP6 is the major protein detected indiagnostic enzyme-linked immunosorbent assays. Beards, G.M., et al., J.Clin. Microbiol, 19:248-254 (1984). Neutralizing and protectiveantibodies are produced to the outer capsid VP7 and VP3 antigens, butinformation on the role of other structural proteins, VP1, 2, 6, and 9,in inducing protection from infection is less clear. Furthermore, othergene products, for example, non-structural proteins (NS 35, 34, 28) arealso synthesized by the rotavirus genome.

The expression vector system is from the insect baculovirus Autographacalifornica nuclear polyhedrosis virus (AcNPV). ACNPV has a genome ofca. 130 kilobases of double-stranded, circular DNA and it is the mostextensively studied baculovirus. Miller, L.K., pp. 203-274 (1981). ACNPVhas a biphasic replication cycle and produces a different form ofinfectious virus during each phase. Between 10 and 24 h postinfection(p.i.), extracellular virus is produced by the budding of nucleocapsidsthrough the cytoplasmic membrane. By 15 to 18 h p.i., nucleocapsids areenveloped within the nucleus and embedded in a paracrystalline proteinmatrix, which is formed from a single major protein called polyhedrin.In infected Spodoptera frugiperda (fall armyworm, Lepidoptera,Noctuidae) cells, AcNPV polyhedrin accumulates to high levels andconstitutes 25% or more of the total protein mass in the cell; it may besynthesized in greater abundance than any other protein in avirus-infected eukaryotic cell.

Polyhedrin is encoded by the virus, and the gene has been mapped andsequenced. The presence or expression of the polyhedrin gene is notrequired for the production of infectious extracellular virus.Inactivation of the polyhedrin gene by deletion or by insertion resultsin mutants that do not produce occlusions in infected cells. Theseocclusion-negative viruses form plaques that are different from plaquesproduced by wild-type viruses, and this distinctive plaque morphology isuseful as a means to screen for recombinant viruses.

SUMMARY OF THE INVENTION

It is therefore, an object of the present invention to produce arecombinant molecule comprising the polyhedrin gene promoter ofbaculovirus Autographa californica nuclear polyhedrosis virus and astructural gene of a rotavirus.

Another object of the present invention is to provide a vaccine againstrotavirus diarrheal disease.

It is a further object of the present invention to form a recombinantmolecule comprising the strong polyhedrin gene promoter and a rotavirusouter capsid protein gene.

Another object of the present invention is to form a recombinantmolecule comprising the strong polyhedrin gene promoter and a rotavirusinner capsid protein gene.

Furthermore, it is an object of the present invention to form arecombinant molecule comprising the strong polyhedrin gene promoter anda rotavirus non-structural protein gene.

It is an additional object of the present invention to form arecombinant molecule comprising the strong polyhedrin gene promoter andmore than one rotavirus gene.

Additionally, it is an object of the present invention to provide asimple assay for rotavirus infection in humans and animals.

Another object of the present invention is to produce antibodies torotavirus.

A further object of the present invention is to provide sufficientprotein to elucidate its structural characteristics.

An additional object of the present invention is to provide sufficientprotein to elucidate its functional properties.

A further object of the present invention is a test kit to facilitatetesting for rotavirus infection in developed and developing countries.

One aspect of the present invention is the provision of a recombinantmolecule, comprising a baculovirus gene promoter, at least one rotavirusgene, the promoter being in spatial relation to the gene so that itregulates the expression of the rotavirus gene. Advantageously, thepromoter is the baculovirus polyhedrin gene promoter and the gene isselected from the group of rotavirus genes consisting of gene 1, gene 2,gene 3, gene 4, gene 5, gene 6, gene 7, gene 8, gene 9, gene 10, gene11, and any combination thereof. Specific embodiments have employed thesimian SA11 genes 6, 9 and 10 to produce VP6, VP7 and NS28 proteinsrespectively. After gene expression, the proteins are isolated in theirnative state.

There is provided in accordance with another aspect of the presentinvention a method of producing the recombinant molecule comprising thesteps of inserting at least one rotavirus gene into a baculovirustransfer vector, then transferring the rotavirus gene in the baculovirustransfer vector to the baculovirus Autographa californica nuclearpolyhedrosis virus genome DNA by cotransfection of Spodoptera frugiperdacells with wild type Autographa californica nuclear polyhedrosis virusDNA, subsequently selecting the recombinant polyhedrinpromoter-rotavirus gene containing the inserted rotavirus DNA moleculeby identifying virus containing occlusion-negative plagues, and finallypurifying the plaques to obtain recombinant molecule virus stocks.Advantageously, the gene is selected from the group of rotavirus genesconsisting of gene 1, gene 2, gene 3, gene 4, gene 5, gene 6, gene 7,gene 8, gene 9, gene 10, gene 11 and any combination thereof.

Furthermore, another aspect of the present invention is that antibodiescan be produced to rotavirus proteins by a method comprising the stepsof synthesizing the rotavirus protein with the recombinant molecules,subsequently innoculating intramuscularly, orally or intraperitoneally ahost animal with the protein to produce an antibody and isolating andpurifying the antibodies thus produced.

Another aspect of the invention is the detection of rotavirus in humanbiological specimens with a method comprising the steps of contactingthe biological specimen with antibodies so that the antibodies androtavirus bind to form an antibody--rotavirus complex and measuring theamount of this complex to determine the amount of rotavirus in thebiological specimen.

In order to facilitate the use of the antibody to measure rotavirusinfection in both developed and developing countries a test kitcomprising the antibody, an appropriate means for collecting andanalyzing the biological sample, has been prepared. The means forcollecting and analyzing in the preferred embodiment include stoolcollection containers, all reagents for diluting and testing the stooland the appropriate container to run the tests.

Additionally, in the present invention a vaccine to rotavirus isproduced from the group of proteins consisting of VP1, VP2, VP3, VP4,VP6, VP7, VP9, NS35, NS34, NS28 and any combination thereof. Theseproteins are synthesized from recombinant molecules. The antigen can bea mixed antigen formed by coexpression of genes of recombinant moleculesor formed by synthesizing individual proteins with subsequent mixing ofthe proteins in vitro.

Immunizing and/or prophylaxis of humans and animals against rotavirusgastrointestinal disease is achieved by parenterally or by orallyadministering an immunological effective dose of the vaccine or a dosewhich will delay the onset and decrease the symptoms of the rotavirusinfection. Additionally, immunization may be achieved by activeimmunization of the vacinee (infant, adult or animal) or through passiveimmunization of the infant or young animal by immunization of the motherprior to birth.

The structure and function of the rotavirus proteins can be determinedby producing sufficient quantities of the rotavirus protein from therecombinant molecules for structural analysis, for functional analysisand for analysis of the immune response to specific viral proteins.Analysis of structure and function will increase our understanding ofthe immunological, biochemical and pathological effects of rotavirusdisease.

The present invention describes the cloning and expression of individualprotein products. Analysis of the antigenic, functional and molecularproperties of the expressed gene products allows examination of theintrinsic and functional properties of each gene product. Thus thepresent invention provides an increased understanding of each individualprotein in the virus structure, preparation and assembly. In addition,the present invention provides for easy and direct dissection of thehumoral and cell mediated immune responses to specific viral proteinsbecause of the availability of high levels of the individual proteins.Furthermore, the large amounts of immunogenic structural proteinsfacilitates vaccine testing and the production of inexpensive diagnostictests.

The present invention describes the formation of recombinant moleculeseach containing a rotavirus gene or genes downstream from a highlyactive promoter gene of the baculovirus Autographa californica nuclearpolyhedrosis virus (AcNPV). This expression vector system provides forsynthesis of the major proteins of the simian rotavirus SA11 in insectcells. This baculovirus expression system efficiently produces the SA11proteins from recombinant molecules containing the promoter gene and theRNA genome segment. This includes the proteins of the outer capsid, theinner capsid and non-specific proteins. The major proteins which havebeen produced by this expression system are VP6, VP7 and NS28. Oneskilled in the art will readily recognize the different classes ofproteins thus produced and will appreciate the applicability of thepresent invention for the expression of all of the rotavirus genes.

Immunological and biochemical analysis indicates that the proteinproduced by the baculovirus vector system possesses both the nativeantigenic determinants and an oligomeric confirmation. One skilled inthe art will recognize that the availability of large amounts of theseproteins facilitates the determination of the intrinsic biochemical andfunctional properties of these proteins in the rotavirus replicationprocesses and viral morphogenesis and increases the immunogenicity ofthese proteins as a vaccine.

The baculovirus possesses several characteristics which make it ideallysuited as an expression vector for cloned eukaryotic genes. Theseinclude:

(1) The ability of this rod-shaped virus to encapsidate its own viralDNA containing large pieces of foreign DNA inserted into thenon-essential polyhedrin gene;

(2) The presence of a very strong promoter which continues to directtranscription late in infection after extracellular virus is producedand after host genes and most viral genes are turned off. Thus theproteins controlled by this promoter are often easily distinguishablefrom host background proteins;

(3) The invertebrate cell cultures used with this vector are not derivedfrom insect vectors that harbor human or animal diseases, are notsusceptible to human pathogens and do not contain any undesirable genes,such as, oncogenes. The use of these cultures thus simplifies the costsof biological safety testing procedures for recombinant DNA productsusing this vector;

(4) The host range of the present strain of the vector is limited tocultured lepidoptera cells;

(5) Since AcNPV is currently being considered for agriculture use as aviral insecticide to control certain lepidoptera and insect pests, allsafety tests required by the Environmental Protection Agency have beencompleted and the vector has been certified for human use;

(6) The recombinant progeny viruses produce plaques that do not containocclusions and thus the engineered recombinants can be easilyidentified;

(7) Recombinant virus stocks of high titer can be easily produced toinfect cells for protein production.

This expression vector has been successfully used to express severalforeign eukaryotic and prokaryotic genes including β-interferon (yields1-5 mg/l) and chloramphenicol acetyl transferase (yields 50-100 mg/l).The proteins synthesized with this vector system have been shown to becorrectly processed, i.e., β-interferon was glycosylated and the aminoterminal signal sequences were correctly removed. The protein products,whether glycosylated or not, have sometimes been secreted from theinfected cells into the media. Since the media components can becontrolled, the purification of the desired gene product from the mediacan be simplified. Large-scale production by fermentation processes ispossible because the invertebrate insect cell, which is used, can begrown in suspension culture.

To be effective against agents that replicate on the mucosal surfaces avaccine should induce the local antibodies required for immuneprotection. Based on this concept, several conventional first generationlive rotavirus vaccines are being developed. Formulations for these livevaccines include the use of attenuated human rotavirus strains, animalrotavirus strains or mixed animal-human viruses. The total number ofvirus groups or virus serotypes that must be included in these livevaccines is undetermined. However, if heterologous viruses are able toproduce protective responses in humans and animals the number may belimited.

The present invention discloses an alternate method of vaccineproduction which avoids the problems associated with live attenuatedviruses. This invention discloses the production of rotavirus specificproteins from cloned genes. The present invention discloses a highlyefficient production of vaccine from rotavirus genes cloned inbaculovirus in infected insect cells. The nonessential structural regionof the baculovirus polyhedrin gene viral genome is deleted and thecloned rotavirus gene(s) are inserted into this region such thatsynthesis is regulated by the viral polyhedrin promoter. One skilled inthe art will quickly recognize that engineered cloned genes in bacteria,yeast, mammalian or insect cells using appropriate expression vectorswill also produce similar gene products. However, the most efficientappears to be baculoviruses. This efficiency is due in part to thehighly active promoter gene at this site.

Thus, the most straightforward and practical approach to developing"second generation vaccines" is to produce rotavirus proteins in aprokaryotic or eukaryotic expression system for direct use as antigens.We have successfully produced proteins from the rotavirus SA11 genes ina baculovirus vector system. The method of the present inventionproduces synthetic polypeptides which are a very cost effective means ofproducing a vaccine for immunization in both the developed and thedeveloping world.

We have found that the nature of the baculovirus, as well as theefficient polyhedrin promoter, result in high level expression of therotavirus major capsid in gene. Furthermore, the baculovirus systemovercomes many of the problems associated with high level expression inbacteria. For example, in most bacteria high level production results ina product that is insoluble. Solubilization of these proteins frombacteria requires further purification. Subsequent administration of theproteins as vaccines often results in a significant loss ofimmunogenicity. In the present system, the strong promoter produces highconcentrations of the antigen which is secreted from the cell into themedia. The antigen is easily purified since the media composition can becontrolled. Therefore, there are minimal problems associated withsolubilization and purification. Additionally the easy purificationallows for the retention of the native protein state.

Further objects, features and advantages will be apparent from thefollowing description of preferred embodiments of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1. Kinetics of protein synthesis of VP6 in infected Sf cells. Theproteins synthesized in mock-(M), wild-type baculovirus-(AcNPV) orbaculovirus recombinant-infected Sf cells were labeled with ³⁵S-methionine (30 uCi/ml) at the indicated times (in hours). The proteinsin cells harvested 2 hours later were then analyzed in 12%polyacrylamide gels. Polyhedrin and SA11 VP6 are indicated by arrowheadsin the left- and right-hand panels, respectively.

FIG. 2. Expression and immunoreactivity of SA11 VP6 in Sf cells. ³⁵S-methionine-labeled proteins synthesized in Wt or SA11 gene 6recombinant infected Sf cells. AcNPV polyhedrin () and SA11 VP6 () arehighlighted. The immunoreactivity of these proteins with antiserum toSA11 particles (αSA11) or with monoclonal antibodies to epitopes present(αSGI, αcom) or absent (αSGI) on SA11 VP6 is shown. The left-hand panelshows reactivity of cell-associated VP6 while the panel on the rightshows reactivity with VP6 frora cells (C) or from the media supernatant(S) from cells infected with two different gene 6 recombinants.

FIG. 3. Detection of VPX expression by ELISA.

ELISA results showing amounts of VP6 detected by ELISA in media () orin SA11-infected Sf cells (▪) at the indicated times postinfection. Theamounts of VP6 at each time-point were quantitated by direct comparisonof the optical density (OD) readings of SA11 6.1-infected cell sampleswith the OD values on a it,3z standard curve of a known amount ofaffinity-purified VP6. Background OD values (0.001-0.016) from mock andwt AcNPV-infected cells were subtracted. The range of the amounts of VP6detected in different infections are also shown.

FIG. 4. Comparison of conformation of expressed and authentic VP6. Thepolypeptide(s) in single-shelled (ss) SA11 particles or inbaculovirus-expressed VP6 were separated on 12% polyacrylamide gelsafter heating either at 100° C. for 2 minutes or at 37° C. for 30minutes in sample buffer in the presence (+) or absencea (-) of2-mercaptoethanol (BME). The gels were processed for fluorographyrandexposed to film to detect radiolabeled proteins or the separatedproteins were electrophoretically transferred to nitrocellulose and thendetected using the SGI monoclonal antibody.

FIG. 5. Characterization of guinea Pig antiserum produced to expressedVP6. ³⁵ S-methionine-labeled polypeptides in SA11-infected MA104 cells(lane 1) were immunoprecipitated with guinea pig antiserum made toconcentrated proteins from wild-type baculovirus (AcNPV)-infected Sfcells (lane 5); VP6 purified from recombinant-infected. Sf cells (lane6); concentrated (but unpurified) proteins from SA11-6-infected Sf cells(lane 7); or purified double-shelled SA11 (lane 8). Lane 9 shows VP6immunoprecipitated with monoclonal antibody to the SGI epitope on VP6.Pre-immune serum from all animals showed no reactivity with any proteinsfrom these lysates (lanes 2-4). Viral proteins VP2, VP3, VP5, VP6 andVP7 that react with antiserum to double-shelled virus are seen in lane8; VP6 is highlighted with an arrow. Lanes 2-9 show analyses performedusing undiluted or a 1:10 dilution of the indicated serum.

FIG. 6A-6C. Assembly of expressed VP6 into structures withcapsomere-like morphology. Expressed VP6 was partially purified and itsstructure was examined by electron microscopy following staining with 2%aqueous uranyl acetate. Tubular structures that were assembled in vitrowere observed. These structures show hexagonal subunit arrangementstypical of the rotavirus inner capsid. Confirmation that these subunitscontain VP6 was shown by specific immunologic reactivity with anti-SGIserum but not with anti-NS28 serum. The antisera had been conjugated tocolloidal gold to facilitate visualization of antibody reactivity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The general method for the successful synthesis of foreign proteins fromcloned genes in a vector system involves the insertion of the foreigngene downstream from the promoter in the expression vector. Thus theproduction of the protein from the cloned gene will be controlledthrough the promoter which directs the transcription of messenger RNA(mRNA).

The procedure involves isolating full length gene clones from rotavirus.These can be isolated from a variety of human or animal virus strains bywell known molecular biology techniques. Cloned CDNA is synthesized fromgenomic double-stranded RNA segments or from mRNA transcribed from thesesegments by the endogenes viral RNA polymerase. First, strand cDNA issynthesized using reverse transcriptase and double-stranded cDNA isobtained either by hybridization of complementary plus and minus strandssynthesized from denatured ds RNA or by second strand sythesis of dCtailed cDNA using reverse transcription or Klenow fragment.Double-stranded CDNA is then tailed with oligo dG and inserted into theP-1 site of a bacterial plasmial such as pBR322 or one of itsderivatives. After amplification of the CDNA, identification of its geneorigin and sequencing to determine it is full-length, the selected geneis subcloned into the baculovirus transfer vector to begin the processto make and select the recombinants. The exact procedures for subcloningeach cloned rotavirus gene will vary depending on the gene sequence. Ingeneral a restriction endonuclease is used to remove the gene from theoriginal bacterial plasmid. This gene segment is then cloned andinserted into the baculovirus transfer vector. Recombinant transfervector DNA is then selected by antibiotic resistance to remove anynon-recombinant plasmid DNA and subsequently amplified and purified fortransfection into Spodoptera frugiperda (SF) cells. Standard viral DNAis used to co-transfect Spodoptera frugiperda (SF) cells. Putativerecombinant viruses containing the recombinant molecules are isolatedfrom the virus yield from these transfected monolayers. Because thepolyhedrin structural gene has been removed, plaques containing therecombinant viruses can be easily identified since they lack occlusionbodies. Confirmation that these recombinants contain the desired gene isestablished by hybridization with specific gene probes.

The expression of rotavirus proteins is determined in SF cells orinsects infected with the virus containing the recombinant DNA. Theinfection process, including viral protein synthesis, viral assembly andpartial cell lysis may be complete by approximately 72 hourspost-infection. This may be protein dependent and thus may occur muchearlier or later. The proteins produced in infected cells areradiolabeled with ³⁵ S-methionine, ³ H-leucine, or ³ H-mannose and bothcell-associated and cell-free polypeptides are analyzed byelectrophoresis on polyacrylamide gels to determine their molecularweight. The expression of these products is examined at different timespost-infection, prior to cell lysis.

Immunological identification of recombinant products is examined byeither direct immunoprecipitation or by Western blots. For Westernblots, cell-associated proteins or the proteins in the media areseparated on SDS polyacrylamide gels, transferred onto nitrocellulosefilters, and identified with antiserum to the rotavirus-specificproteins or to the polyhedrin. Specifically bound antibody is detectedby incubating the filters with ¹²⁵ I-labeled protein A or enzymeconjugated anti-antibody, and followed by exposure to X-ray film at -80°with intensifying screens or colorimetic reaction with enzyme substrate.

Alternatively, extracts of cell-associated and soluble proteins are usedfor immunoprecipitation with monoclonal antibodies or with monospecificor polyclonal antivirus serum, to determine whether rotavirus proteinsare being produced. These analyses determine whether the proteinsproduced in the recombinant-infected insect cells are identical in sizewith those produced in cell-free translation systems or in rotavirusinfected-cells. Finally, the amounts of each protein are quantitatedbefore and following protein purification.

The product(s) produced by the rotavirus gene are similarly analyzed todetermine if they are properly processed (i.e., that the signal peptidesare cleaved and whether they are correctly glycosylated).

Once recombinant vectors that express the proteins are established, thenthe cells are infected with different combinations of the recombinants.These viral proteins should form capsid subunits and be released fromthe infected cells as a subunit. Studies on the morphogenesis andimmunologic properties of rotavirus particles will allow the predictionof how viral subunits are assembled in infected cells or in cell-freesystems.

The next step is to purify the proteins for use and evaluation assubunit vaccines. In this expression system the rotavirus proteins areoften released into the medium. Media from these infected cells isconcentrated and the proteins purified using standard methods. Saltprecipitation, sucrose gradient centrifugation and chromatography, highor fast pressure liquid chromatography (HPLC or FPLC) is used becausethese methods allow rapid, quantitative and large scale purification ofproteins, and do not denature expressed products. Thus, we achieve thehighest probability of producing proteins that retain their antigenicproperties.

The efficiency of synthesis of the desired gene product is dependent onmultiple factors including: (1) the choice of an expression vectorsystem; (2) the number of gene copies that will be available in thecells as templates for the production of mRNA; (3) the promoterstrength; (4) the stability and structure of the mRNA; (5) the efficientbinding of ribosomes for the initiation or translation; (6) theproperties of the protein product, such as, the stability of the geneproduct or lethality of the product to the host cells; (7) the abilityof the system to synthesize and export the protein from the cells, thussimplifying subsequent analysis, purification and use.

Production of the viral proteins in high yields from expression vectorsystems provides a novel way to study viral protein function and todevelop diagnostic tests and vaccines. This invention discloses and isdirected to an evaluation of the antigenic and molecular properties ofthe simian rotavirus SA11 major capsid protein VP6 and glycoprotein,VP7, as well as the non-structural protein NS28 produced using thebaculovirus expression system.

Several properties and advantages of the present invention employingexpressed rotavirus proteins will be readily obvious to one skilled inthe art. For example, (1) VP6 is not a glycoprotein and is not known tobe found in the media from SA11-infected mammalian cells, however, it ispresent in the media from the baculovirus recombinant-infected cells.Although it is not clear whether VP6 is actively secreted into the mediaor is released as a result of cell lysis, the property of highconcentration in the media greatly facilitates the purification of VP6.Other non-glycosylated proteins, β-galactosidase and chloramphenicoltransferase, also are found in the media after production with the samebaculovirus expression vector. (2) On the other hand, β-interferon isglycosylated and is also found in the media of recombinant-infectedcells. Thus a variety of protein classes can be found in the media,significantly simplifying isolation and purification. (3) Reactivitywith available monoclonal antibodies suggested that nativeimmunoreactive determinants were conserved when VP6, VP7 and NS28 weresynthesized using the baculovirus expression vector. Additionally, thedemonstration that the expressed and purified VP6 protein showsproperties of an oligomer, the reported structure of VP6 invirus-infected cells or in virus particles, confirms that the nativeconformation is maintained in the vector system synthesis. This oligomerformation is associated with disulfide bonds and is an intrinsicproperty of VP6.

A variety of cultivatable and non-cultivatable strains of rotavirus areknown. These strains are known to cause disease in humans and animals.Vaccines against these different strains thus will be useful incombating disease in humans, agricultural animals and pet animals.Examples of these strains in different virus groups are: Simian SA11 andMMU18006; canine CU-1; equine H2 and H1; porcine Gottfried, SB-1, SB-2,OSU, EE and A580; bovine NCDV, UK, B641, B720, B14, II-2 and B223;Turkey Ty1 and Ty3; chicken Ch1; human Wa, KU, K8, DB, DS-1, S2, KUN,HN-126, 390,M, P, YO, 14, 15, McM2, MO, Ito, Nemoto, St. Thomas 4,Hochi, Hosokawa, adult diarrhea rotavirus (ADRV), PaRV and BRVLA. Estes,M.K. et al., "Antigenic Structure of Rotavirus" in Immunochemistry ofViruses. Elsevier p. 389-405 (1985), The disclosure of which is herebyincorporated by reference and; Nakata, S. et al., J. Infectious Diseases154:448-455 (1986) the disclosure of which is hereby incorporated byreference. Any strain can be used to incorporate the genes into vectorsto make recombinant molecules.

SPECIFIC EXAMPLES MATERIALS AND METHODS

Cells and virus: The simian rotavirus SA11 was grown in cultures ofMA104 cells as described in Estes, M. K. et al. J. Virol. 31:810-815,1979, the disclosure of which is hereby incorporated by reference. Thebaculovirus Autographa californica (AcNPV) and the recombinant viruspAc461/SA11-6 were used to infect Spodoptera frugiperda (Sf)(IPLB-SF21-AE) cells at a multiplicity of infection (MOI) of 10plaque-forming units per cell. Sf cells were grown and maintained onHink's medium containing 10% fetal bovine serum (FBS) as described inSmith, G.E. et al. J. Virol. 46:584-593, 1983, the disclosure of whichis hereby incorporated by reference.

Construction and selection of baculovirus recombinants

Gene 6: SA11 gene 6 was originally in the PstI site of pBR322. Theresulting clone, pSA11-6, was digested with AhaIII and HpaII andsubcloned into the SmaI site of the baculovirus transfer vector pAc461.The plasmed pAC461 was derived from pAc311 by cleavage with BamHI andkpnI, digestion with Ba131 nuclease, treatment with DNA polymerare I(Klenow fragment) to produce blunt ends, ligation of a SmaI GCCCGGGG!linker, cleavage with SmaI and religation. The plasmid pAC461 has adeletion between positions -7 and +670 in the baculovirus polyhedringene. After transfection into Escherichia coli, plasmids in recombinantampicillin-resistant colonies were screened by restriction enzymeanalysis for inserts in the correct transcriptional orientation, and onewas designated pAc461/SA11-6. pAc461/SA11-6 is missing the first sevennucleotides of the 5' end of SA11 gene 6 and has about 70 extra basepairs that were added at the 3' end during the original cloning intopBR322. Transfer of SA11 gene 6 cDNA from this vector to the AcNPVgenome DNA was achieved by cotransfection of Sf cells with wild-typeAcNPV DNA using the calcium phosphate precipitation procedure asdescribed in Smith, G.E., et al. J. Virol 46:584-593, 1983. The ACNPVDNA (1 μg) was mixed with 2 μg of pAc461/SA11-6, and brought to 950 ulwith HERBS/CT 0.137M NaCl, 6 mM D-glucose, 5 mM KCl, 0.7 mM Na₂ HPO₄ 0.7H₂ O, 20 mM HEPES, 15 μg/ml sonicated calf thymus (CT) DNA, pH 7.1!, andvortexed. While the mixture was being slowly vortexed, 50 ul of 2.5MCaCl₂ was added and a precipitate was allowed to form at roomtemperature for 30 minutes. The precipitated DNA was added to 2 ml ofHink's medium supplemented for 10% FBS in a 25-cm² flask seeded with2.5×10⁶ Sf cells. Following incubation at 27° C. for 4 hours, the mediumwas removed, the monolayer was washed with fresh Hink's mediumcontaining 10% FBS, and, after the addition of 5 ml of Hink's mediumsupplemented with 10% FBS, the flask was incubated at 27° C. The cellswere observed using an inverted microscope for signs of infection and,at an advanced stage of infection (day 5), extracellular virus washarvested and plaqued on monolayers of Sf cells. Recombinants, in whichthe polyhedrin gene had been replaced by the polyhedrin-SA11-6 transfervector DNA by homologous recombination, were selected by identifyingocclusion negative plaques with an inverted phase microscope asdescribed in Smith, G.E. et al. J. Virol. 46:584-593, 1983. Virus inocclusion-negative plaques was plaque purified three times and used topropagate virus stocks.

Gene 9: Clones of SA11 gene 9 were constructed using the same proceduresas for gene 6 clones (described in Estes, M. K. et al. Nucl. Acid Res.12:1875-1887, 1984, the disclosure of which is incorporated byreference). These clones were selected from the library of cDNAs usingprobes for gene 9. The identification of these clones was confirmed byproduction of the specific protein in cell-free translation systemsderived from rabbit reticulocytes and wheat germ extracts. Suchtranslation reactions were programmed with mRNA selected byhybridization to the gene 9 cDNA immobilized on filters.

The entire coding sequence of each of these clones was then inserted inthe baculovirus transfer vector prior to transfection of Sf cells toobtain baculovirus recombinants containing SA11 gene 9 DNA. For gene 9constructions, the gene 9 cDNA was excised from pBR322 with Pstl andthis DNA was gel purified and inserted into the Pstl site of the pAC461baculovirus transfer vector. Another alternative is that prior toinsertion into pAC461 the tails or other sequences may be modified ordeleted. Gene 9 stocks were then obtained following the procedures usedfor gene 6.

Gene 10: Clones of SA11 gene 10 were constructed using the procedure ofBoth G. W. et al., J. Virol. 48:335-339 (1983). These clones wereselected from the library of cDNAs using probes for gene 10. Theidentification of these clones was confirmed by production of thespecific protein in cell-free translation systems derived from rabbitreticulocytes and wheat germ extracts. Such translation reactions wereprogrammed with mRNA selected by hybridization to the gene 10 cDNAimmobilized on filters. The entire coding sequence of each of theseclones was then inserted in the baculovirus transfer vector prior totransfection of Sf cells to obtain baculovirus recombinants containingSA11 gene 10 DNA. For gene 10 constructions, the gene 10 cDNA wasexcised from pBR322 with AMAIII and HpaIII as described for gene 6 andgene 10 was inserted into the SmaI site of the pAC461 baculovirustransfer vector. Gene 10 stocks were then obtained following theprocedure used for gene 6.

Radiolabeling analysis of proteins synthesized in infected Sf cells. Sfcells infected with recombinant SA11-6 or recombinant SA11-9 orrecombinant SA11-10 or with wild-type AcNPV were radiolabeled atdifferent times post-infection with 15 μCi/ml of ³⁵ S-methionine inmethionine-free Hink's medium supplemented with 10% FBS. The cells andmedia were harvested at 42 hours post-infection, and the cells werepelleted at 1400×g at 4° C. for 5 minutes. The cell-free supernatant wasremoved and saved for testing, and the cell pellet was suspended in RIPAbuffer 0.15M NaCl, 0.01M Tris-HCl (pH 7.2), 1% trasylol, 1% soldiumdeoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS)!.Proteins were immunoprecipitated from these samples as described inChan, W. K. et al. Virology 151:243-252, 1986, the disclosure of whichis incorporated by reference. Antisera used for immunoprecipitation werea polyclonal guinea pig anti-SA11 serum (gp α SA11) that reacts withSA11 structural proteins VP 2, 3, 6 and 7; ascites fluids from miceinjected with hybridoma cells that secrete monoclonal antibodies to VP6(gene 6) subgroup I (α SGI) or subgroup II (α SGII) or to a commondeterminant on VP6 (α common); monoclonal or polyclonal antibodies toVP7 (gene 9); or monoclonal antibodies to NS28 (gene 10). In some casesthe immunoreactivity of the expressed protein was also analyzed onimmunoblots.

partial purification of the expressed rotavirus proteins (VP6. VP7 andNS28): For example, gene 6 recombinant-infected Sf cells were labeled 28hours post-infection with ³⁵ S-methionine (30 μCi/ml, 1200 Ci/m moleAmersham Corp.) in Hink's medium lacking FBS. The cells and media wereharvested separately at different times post-infection by scraping cellsinto the media and pelleting the cells by centrifugation at 1000 RPM for20 minutes at 4° . Proteins can be purified from the media orcell-associated material. For example from the media followingclarification by centrifugation, 100,000×g for 30 minutes, and thenapplying the supernatant to a 5 to 20% continuous sucrose gradient for23 hours at 100,000×g. Fractions containing the expressed proteins thatlacked contaminating bovine serum albumin were pooled, dialyzed against10 mM Tris-HCl (pH 7.5), and used either directly or afterlyophilization.

Additionally, wild-type AcNPV and recombinant expressed proteins wereprepared by the same infection conditions and harvesting procedure, butnot purified further. In this case, medium from infected cells wascollected, dialyzed against 10 mM Tris-HCl (pH 7.5), and used directlyor lyophilized. Concentrations of proteins were approximated bycomparing the amounts of protein to quantitative marker proteins afterelectrophoresis on 12% polyacrylamide gels and staining with silvernitrate or Coomassie blue or by a quantitative ELISA.

Affinity purification of expressed proteins: Protein A-Sepharose CL-4Baffinity columns (Pharmacia Inc.) were prepared by crosslinkingmonoclonal antibody supernatant to the gel using dimethylpimelimadedihydrochloride. Unlinked antibody was removed by washing with 0.1Mborate buffer (pH 8.3). Supernatants from Sf cells infected with theappropriate recombinant genes were mixed with the immunomatrix, shakenat room temperature, and centrifuged at 1500 RPM for 1 minute. Theimmunomatrix gel was then suspended in borate buffer and poured into adisposable Econocolumn (Bio Rad). The column was washed sequentiallywith 50 ml each of buffer A 0.5M NaCl, 0.05M Tris-HCl (pH 8.2), 1mMEDTA, 0.5% Nonidet P-40!, buffer B (0.15M NaCl, 0.05M Tris-HCl (pH 8.2),0.5% Nonidet P-40, 0.1% SDS!, and buffer C (0.15M NaCl, 0.5% sodiumdeoxycholate) with borate buffer washes between each. After a final washwith borate buffer, the protein was eluted from the gel with 0.1Mglycine-HCl (pH 2.5) into tubes containing 2M Tris base to neutralizethe acid. The eluted protein was analyzed by electrophoresis on 12%polyacrylamide gels and was used as a standard in ELISA to quantitatethe kinetics and levels of expression of the proteins VP6, VP7 or NS28.For example and not limitation subgroup I (α SG1) monoclonal antibodycan be used to purify VP6. One skilled in the art will readily recognizethat specific monoclonal antibodies to other rotavirus proteins can beused to purify these proteins..

Production of antiserum to expressed Proteins: Guinea pigs (Elm HillBreeding Farms, Chelmsford, Mass.) shown to lack rotavirus antibodieswere used for the production of antiserum to expressed VP6 protein. Eachguinea pig was inoculated intramuscularly twice at 3-week intervals asdescribed in Estes, M. K. et al. J. Virol. 31:810-815, 1979. Theantigens used were partially purified expressed VP6, or proteinsconcentrated from the supernatant from Sf cells infected with eitherAcNPV or pAc461/SA11-6. The guinea pigs were bled 1 week after thesecond injection of antigen, and the serum samples were tested for thepresence of antibodies by immunofluorescence, immunoprecipitation, ELISAand immune electron microscopy assays. VP6 is shown for example andsimilar methods are used to produce antiserum to other rotavirusproteins including VP7 and NS28.

SDS-Polvacrylamide gel electrophoresis: Protein products were analyzedby polyacrylamide slab gel electrophoresis using a modification of themethod of Laemmli with 12% separating and 4% stacking gels as describedin Chan, W. K. et al. Virology 151:243-252, 1986. Beforeelectrophoresis, unless otherwise stated, samples were dissociated byboiling for 3 minutes in sample buffer containing 1% SDS, 10%2-mercaptoethanol, 0.5M urea, 0.05M Tris HC1 (pH 6.8), 10% glycerol, and0.0025% phenol red. Radiolabeled proteins were monitored on gelsfollowing fluorography, as described in Mason, B. B. et al. J. Virol.46:413-423, 1983, the disclosure of which is incorporated by reference.

Expression of SA11 proteins in Sf cells. Three baculovirus recombinantscontaining an SA11 gene 6 CDNA insert were initially identified andtested for their ability to produce SA11 VP6 after infection of Sfcells. The ³⁵ S-methionine containing polypeptides in cells infectedwith AcNPV or SA11 gene 6 recombinant are shown in FIG. 1. A new band atthe expected molecular weight of 41,000 (41K) was seen inrecombinant-infected cells while the polyhedrin protein was only seen inAcNPV-infected cells. The kinetics of synthesis of this proteindemonstrated that the radiolabeled VP6 is initially detected at 22 hourspost-infection, and its synthesis is still detectable at 105 hourspost-infection. One skilled in the art will recognize the advantages ofthis late synthesis for identification and isolation of foreign protein(rotavirus) from early synthesized protein (baculovirus). Protein VP6 isshown for example only and not limitation. Other gene products ofrotavirus can be similarly expressed and detected in Sf cells.

Immunoreactivity and localization of expressed proteins: Identificationof the 41K band as authentic VP6 was shown by positive immunoreactivitywith polyclonal antisera prepared to SA11 virus and with monoclonalantibodies to two epitopes (common and subgroup I) on the SA11 VP6 (FIG.2).

Specificity of these reactions was shown by a lack of reactivity of thesubgroup II antibody with the SA11 41K protein and by finding that noproteins in AcNPV-infected cells reacted with any of these areas.Similar results were found with all three VP6 recombinants.

VP6 was observed in the cytoplasm of recombinant-infected cells byimmunofluorescence, and it could be immunoprecipitated from bothclarified supernatants and cell-associated proteins (FIG. 2). The amountof protein being synthesized can be quantitated by analysis of dilutionsof immunoprecipitated VP6. FIG. 3 shows quantitation with ELISAstandardized with VP6 purified to radiochemical and biochemicalhomogeneity from an immunomatrix column and demonstrates thatimmunoreactive VP6 could be detected as early as 8 hours post-infection.Furthermore the amount of VP6 found in the medium increased with time.The cell-associated VP6 was produced in infected cells in amounts of20-150 μg from a density of 10⁶ cells/ml, and these yields in clarifiedmedia are sufficient for further biochemical and immunologic studiesusing purified VP6. The yield of VP6 is maximized when cells were grownin complete media containing 10% FBS.

Conformational proverties of expressed VP6. It is known that VP6 removedfrom purified bovine rotaviruses or analyzed in infected cells behavesas an oligomeric, possibly trimeric, protein. The VP6 synthesized in thepresent invention possessed conformational properties similar toauthentic VP6. VP6 purified from the media form infected cells possesseda native oligomeric structure, was immunogenic in guinea pigs, and wasable to spontaneously assemble into morphologic subunits. FIG. 4 showsthat the profile of ³⁵ S-methionine-labeled proteins in purifiedsingle-shelled virus after electrophoresis in SDS-polyacrylamide gelswas different depending on whether or not the virus was heated prior toloading the gels. In the presence of heating, characteristic bandsrepresenting VP1 (125K), VP2 (94K), and VP6 (41K) were seen. In theabsence of heating, the VP6 band disappeared and a new band migratingabove VP1 was observed. Similar analyses with baculovirus-expressed VP6(partially purified on sucrose gradients) and withimmunoaffinity-purified VP6 showed that these proteins also formedoligomers (FIG. 4). In the absence of reducing agents(2-mercaptoethanol), an additional band that also migrated as a multipleoligomeric form was, observed. Monomeric and oligomeric forms ofexpresses VP6 were also seen using immunoblots. One skilled in the artwill recognize that the immunoprecipitation studies with monoclonalantibodies and the conformational studies confirm that the expressed VP6possesses a native conformation and furthermore demonstrates thatoligomer formation is an intrinsic property of VP6. FIG. 6 showselectron micrographs of the expressed VP6 that assembled spontaneouslyinto morphological structures. These tubular structures that areexpressed VP6 show hexagonal subunit arrangements typical of therotavirus inner capsid. FIG. 6B shows the VP6 structures labeled withSGI-gold complex. FIG. 6C shows the VP6 structure reacted withα-NS28-gold complex and no specific binding is observed. This shows thespecificity of the tubular structures assembled from VP6.

Characterization of antisera produced to expressed Protein: Antiserawere produced in guinea pigs to concentrated wild-type AcNPV-infected Sfcell proteins, to concentrated gene 6 recombinant-expressed proteins inSf cells, and to VP6 partially purified from the media of infected Sfcells by sucrose gradient centrifugation. The ability of each antiserumto recognize VP6 was tested by immunoprecipitation. In FIG. 5 it can beseen that each pre-immune serum and the antiserum to the AcNPV-infectedSf proteins did not react with any cellular or viral proteins in theSA11-infected MA104 cell lysates, while antisera to the unpurified andto the partially purified VP6 reacted specifically with VP6. Theseantisera were further characterized for their ability to detectrotavirus. The anti-VP6 sera detected rotavirus strains representingeach known human serotype and subgroup with both immunofluorescence andELISA assays.

Vaccination with baculovirus expressed protein: Mouse dams werevaccinated with parenteral immunization with VP6. The regimen includedthree doses, each containing 20 μg VP6 and an adjuvant. As controls,dams were immunized with double-shelled virus, single-shelled virus oradjuvant alone. All pups born to these dams were challenged withrotavirus. Pups whose dam was immunized with double shelled-virus weretotally protected from illness. Pups showed partial protection if theirdams had been immunized with VP6 or single-shelled virus. These pupsexperienced both a delayed onset of illness and less severe disease.Pups born to dams only immunized with adjuvant showed rapid onset andsevere disease. One skilled in the art will recognize the immunizationqualities of VP6. Its pattern suggests that complete protection mayrequire both inner and outer coat antigens. Thus one skilled in the artwill further recognize that mixtures of VP6 and VP7 should provideadequate immunization. As other rotavirus proteins are synthesized,additional mixtures can be formed to provide complete immunizationagainst rotavirus infection. One skillet in the art will recognize thatseveral approaches can be used to form mixtures.

For example, these can include the simultaneous infection of Sf cellswith more than one recombinant, whereby the intrinsic properties of VP6and VP7 can foster the formation of aggregates naturally. Experimentswith the proteins in Sf cells co-infected with VP6 and VP7 suggestoligomers of these proteins are formed. Since VP6 expressed alone ismade as an oligomer and forms morphologic subunits in vitro one skilledin the art will recognize that the co-expressed VP6 and VP7 willnaturally form aggregates or subviral particles. Since VP7 is reportedto be the cell attachment protein, the expression of these epitopes onthe aggregates may enhance the ability of the subunit vaccines to bindto cells and to be a potent immunogen. The co-expression of variouscombinations of rotavirus gene proteins for example VP6, VP7 VP3, NS28and others in Sf should result in natural aggregates and thus theproduction of mixed antigens for immunization in human populations.

Alternatively the rotavirus proteins can be synthesized individually inSf cells and then a vaccine may be formed by mixing the purifiedproteins in vitro. Additionally the immungenicity can be improved overstraight mixtures by solubilization before or during mixing.Solubilization of the proteins allows in vitro aggregation of the mixedantigens. The aggregates should be better immunogenes than simplemixtures of purified proteins since they simulate the natural state.

Since other modes of mixing including co-synthesis and co-expression canbe equally utilized to achieve a mixed antigen immunization vaccine, theabove are for example only.

VP6 was immunogenic in that it induced antisera in guinea pigs that washigh titer when tested by ELISA or immunofluorescence assay. However,the anti-VP6 sera by itself did not neutralize SA11 infectivity whentested in plaque-reduction neutralization assays using 50 plaque-formingunits of virus. Thus there is evidence that even though some in vitrotests do not recognize the immunogenicity of the VP6 protein, actualimmunization in vivo can be achieved (mouse data). The inability of theexpressed VP6 to induce neutralizing antibodies does not rule out apossible role for this protein in inducing protection from infection ordisease, as our understanding of the immunologic and non-immunologicmechanisms that are critical to induce protection from rotavirusinfection and disease remains incomplete. VP6 may be important instimulating cell-mediated immune responses that induce reportedheterotypic immunity in a similar fashion to that of the proteins ofinfluenza virus. Expressed VP6 may also be a useful component in subunitvaccines containing additional expressed outer capsid proteins.

Production of Diagnostic Kits for Rotavirus Detection: Antiserumsproduced to the baculovirus expressed proteins can be used to producediagnostic kits to detect either rotavirus antigen or antibody responsesto rotaviruses. One skilled in the art will recognize that a variety ofdifferent formats can be used to produce these diagnostic kits. Forantigen detection, antiserum made to one or more of the baculovirusexpressed rotavirus proteins is used to detect virus particles orsoluble antigen found in clinical specimens. Fecal specimens arepreferred since the infection is in the gastrointestinal tract.Antiserum made to VP6 is broadly reactive and detects human and animalrotaviruses. Such kits can also use more than one antiserum made towhole virus particles or to other baculovirus expressed proteins tocapture viral antigens. For antibody detection, the antiserum made tothe baculovirus expressed protein can be used to capture viral antigenwhich is then used to detect antibody in human or animal serum samples.

Individually expressed VP6 formed oligomers, and electron microscopicanalysis also revealed further assembly of the expressed trimeric VP6molecules into morphologic subunits that are characteristic of thosefound on single-capsid particles. These subunits have been seenfollowing selective removal of VP6 from the inner capsid of virusparticles. Therefore, protein-protein interactions with other viralproteins or with preformed subviral structures may not be required forsubunit formation. The availability of large amounts of purified capsidproteins will now facilitate dissection of the factors that controlparticle morphogenesis and will allow self-assembly of virus capsids.The native conformation of the present invention is consistent with theuse and advantages of this system to produce eukaryotic proteins.

One skilled in the art will recognize other uses of this expressedprotein to improve current diagnostic tests for rotaviruses or as avaccine. Antiserum made to unpurified or partially purified VP6 detectedcommon antigenic determinants shared by all group A rotaviruses byimmunofluorescence and ELISA tests. Since VP6 is the major proteindetected in available commercial diagnostic tests based on eithermonoclonal or polyclonal antiserum, it is readily apparent to oneskilled in the art that future production of such reagents usingbaculovirus-expressed VP6 becomes cost-effective due to the high yieldsand the ability to produce this protein from infected cells grown inspinner cultures, monolayer or fermentation.

While presently preferred embodiments and examples of the invention havebeen given for the purposes of disclosure, changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention or defined by the scope of the appendedclaims.

What is claimed is:
 1. A recombinant molecule, comprising:(a) abaculovirus gene promoter; (b) a rotavirus gene which codes forrotavirus protein NS28, said promoter spatially positioned in relationto said gene effective to regulate the expression of said gene.
 2. Arecombinant molecule according to claim 1,wherein said baculovirus genepromoter is a polyhedrin gene promoter from a baculovirus Autographacalifornica nuclear polyhedrosis virus.
 3. A method of producing arecombinant molecule comprising the steps of:(a) inserting a rotavirusgene, coding for rotavirus protein NS28, into a baculovirus transfervector; (b) transferring said rotavirus gene in the baculovirus transfervector to the baculovirus Autographa californica nuclear polyhedrosisvirus genome DNA by cotransfection of Spodoptera frugiperda with wildtype Autographa californica nuclear polyhedrosis virus DNA; (c)selecting the recombinant polyhedrin promoter-rotavirus gene molecule byidentifying occlusion-negative plaques, or by hybridization withspecific gene probes or by both procedures; (d) purifying said plaquesto obtain virus stocks containing recombinant molecule.
 4. A recombinantbaculovirus, comprising in its genome:(a) a baculovirus gene promoter,and (b) a rotavirus gene which codes for rotavirus protein NS28, saidpromoter spatially positioned in relation to said gene effective toregulate the expression of said gene.
 5. The recombinant baculovirus ofclaim 4, wherein said baculovirus gene promoter is a polyhedrin genepromoter from a baculovirus Autographica californica nuclearpolyhedrosis virus.
 6. A method of producing rotavirus protein NS28which comprises infecting a host cell with the recombinant baculovirusof claim 4 and culturing the host cell under conditions which permit theproduction of said rotavirus protein NS28 by the host cell.
 7. A methodof producing rotavirus protein NS28 which comprises infecting a hostcell with the recombinant baculovirus of claim 5 and culturing the hostcell under conditions which permit the production of said rotavirusprotein NS28 by the host cell.